Abstract:

The isolation and identification based on phenotypic and phylogenetic characteristics of the thermophilic bacteria from different geothermal
springs (with temperature 27.5–70 °C) distributed on the territory of Armenia and Nagorno Karabakh were carried out. In total 135
thermophilic and thermotolerant bacilli strains were isolated under aerobic conditions at 55-65 °C and indentified based on 16S rRNA
gene sequence analysis as representatives of genera Anoxybacillus, Bacillus, Brevibacillus, Geobacillus, Paenibacillus, Sporosarcina,
Ureibacillus and Thermoactinomyces. These thermophilic bacilli were tested for enzyme production capacities such as lipase, protease,
amylases and biotechnologically valuable enzyme producers were selected.

Thermostable enzymes, which have been isolated mainly from
thermophilic microorganisms, have found a number of commercial
applications as they possess thermal stability to harsh industrial
processes at high temperatures (DeCastro et al., 2016). The
elevation of temperature is accompanied by a decrease in viscosity
and an increase in the diffusion coefficient of organic compounds.
Furthermore, by performing biological processes at high temperature
the risk of contamination is reduced and controlled processes
under strict conditions can be carried out (Antranikian, 2008;
Kurosawa, 2013).

Thermophilic microorganisms are not grouped into a separate
taxonomic unit, but appear in various taxonomic groups and at
various phylogenetic distances throughout the taxonomic system
(Horikoshi and Bull, 2011; Sharma et al., 2013). It was shown that
representatives of the genus Bacillus and related genera to be the
thermophilic aerobes most frequently iso¬lated from terrestrial
geothermal water environments (Sharp et al., 1992). Typically, enzymes
production in the course of Bacillus fermentation processes
occurs during a relatively short period of time, with very low cost
carbon and nitrogen sources. A great part of industrially valuable
enzymes are mainly produced by bacilli (Schallmey et al., 2004).
Therefore, screening for novel biocatalysts from thermophiles
has become a very important field and can open a new horizon
in biotechnology. Between not well known ecological zones of
the Earth, thermal springs located in the Minor Caucasus still represent
a challenge for searching of undescribed biotechnological
resource. The geology of the region where Armenia and NagornoKarabakh
are situated is complex, owing to accretion of terrains
through plate-tectonic processes, and to ongoing tectonic activity
and volcanism (Henneberger et al., 2000; Badalyan, 2000) Numerous
geothermal springs of different geotectonic origin and with
different physicochemical properties are found on the territory of Armenia and Nagorno-Karabakh (Mkrtchyan, 1969). Recently microbiological
investigations of some Armenian geothermal springs
were carry out based on culture independent and molecular methods
(Panosyan, 2010, Hedlund et al., 2013; Panosyan and Birkeland,
2014). Despite this progress very little is known about the diversity
of biotechnologically valuable enzyme producers thriving
in Armenian geothermal springs (Shahinyan et al., 2017).
The present study focuses on isolation and identification of thermophilic
bacilli from geothermal springs of Armenia and Nagorno-
Karabakh as well as on screening of hydrolytic enzymes
(amylase, lipase and protease) producers.

Materials and methods
Study sites and sampling

The location of geothermal mineral springs was determined using
JPS. The samples were collected from Armenian (Akhurik,
Arzakan, Bjni, Hankavan, Jermaxbjur, Jermuk, Tatev, Uys, Karvachar,
Zuar) and Nagorno-Karabakhian (Karvachar, Zuar) geothermal
springs of different geotectonic origin and with different
physicochemical properties (Fig. 1). Temperature, pH and conductivity
were determined in situ using a portable combined pH/EC/
TDS Temperature tester (HANNA HI98129/HI98130). Water, mat
and adjacent sediment samples were collected using sterile glass
flasks and were maintained on ice until processed.

Enrichment and isolation

To enrich aerobic endospore-forming thermophilic bacteria slurry
water, mat and sediment samples (1g) were inoculated in Nutrient
Broth (HiMedia) and incubated overnight at 55, 60 and 65 ºC with
shaking at 150 rpm. Before inoculation all samples were treated at
80 oC for 10 min aiming to isolate only spore-forming microorganism
(Netrusov, 2005). Cultures showing different colony morphology
was further purified by streaking samples on the same
medium supplemented with agar (2 %, w/v). All colonies obtained
on plates were picked and purified by streaking onto same medium
at least three times. The subcultures were considered pure after microscopic
observation of a single morphological type per culture.
The subcultures’ purity, cell morphology and motility, endospore
location and were determined by phase-contrast microscopy of
freshly prepared wet mounts.

Phenotypic characterization of isolate. All isolates were tested for
their colony morphology, Gram reaction and thermophilic growth
using common methods (Netrusov, 2005). The temperature range
for growth was determined after incubation of isolates at 5 to 80 ºC
by 5 ºC intervals. The pH dependence of growth was tested at pH
range of 5 to 12. The range of NaCl concentrations for growth was
determined by adding 0 to 15 % NaCl to the incubation medium.
Catalase activity was determined by bubble formation in a 3 %
hydrogen peroxide solution. Biochemical tests such as nitrate reduction,
gas production from D-glucose, formation of dihydroxyacetone
and Voges-Proskauer test were carried out according to (Gordon et al., 1973).

DNA extraction, polymerase chain reaction (PCR) and sequencing

DNA was extracted from pure isolates using GenEluteTM Bacterial
Genomic DNA Kit (Sigma) according to the manufacturer’s
recommendations and used as a template in the PCR assays.
16S rRNA genes were amplified using universal primer pairs 27f
(5’-GAGTTTGATCCTGGCTCA-3’) and 1525r (5’-GAAAGGAGGAGATCCAGCC-3’)
(Escherichia coli numbering). PCR
mixtures used for amplification of sequences contained 10 ng
DNA, 5 µl 10 X PCR buffer, 5 µl 10 mM dNTP (dATP, dGTP,
dCTP and dTTP), 1 µl each primer (25 pmol/µl), 1,5 mM MgCl2,
0,2 µl Taq DNA polymerase, and sterile water up to the final
vo¬lume of 50 µl. PCR amplification was completed using an
DNA Engine thermocycler (BIO RAD). First, the templates were
denaturized for 3 min at 96 ºC, then 30 cycles of the following
steps were completed: denaturation for 30 at 96 ºC for, annealing
for 30 s at 55 ºC, and extension at 2.5 min at 72 ºC. The 30 cycles
were followed by a final 10 min extension at 72 ºC. PCR proucts
were viewed under UV light after standard ethidium bromide gel
electrophoresis. PCR products were purified with GenElute™
PCR Cleanup Kit (Sigma). Sequencing of bacterial 16S rDNA
amplicons were performed on ABI PRISM capillary sequencer according
to the protocol of the ABI Prism Big-Dye Terminator kit
(Perkin Elmer) using above mentioned primers. Raw data of DNA
sequences was analysed with Chromas and BioEdit software. A
nucleotide BLAST search was performed in order to obtain information
on the phylogenetically closest relative (National Center
for Biotechnology Information, http://www.ncbi.nlm.nih.gov/
Blast) (Altschul et al., 1997).

Results and Discussion

The temperature of water of studied geothermal springs in the outlet varied between 27-70 oC. Despite the varying phy¬sical-chemical
properties of the thermal springs used for the sampling, they belong to the category of hot springs from low-tem¬perature fields and are
characterized by neutral to alkaline pH and high concentration of dissolved minerals and gases. Studied mesothermal springs mainly
are related to the hydrocarbonate sodium or hydrocarbonate-sulphate sodium-mag¬nesium classes. The location and physical-chemical
parameters of geothermal springs of Armenia and Nagorno-Karabakh are shown on the Table 1.

Collected 52 water, mat and adjacent sediment samples were analyzed to evaluate the total thermophilic aerobic endospore-forming
bacterial abundance. A total of 135 isolates with different colony morphologies were obtained from the samples from different geothermal
regions. All isolates were rod-shaped, gram-positive, endospore-forming, catalase-positive bacteria. Results of nitrate reduction, gas
production from glucose, Voges-Proskauer test and some other properties are shown on the Table 2.

Isolates optimal growth temperature of which was between 60-65 oC were defined as oligate thermophilic, whereas the ones growing
at 55 oC were taken as thermotolerant. Although 135 of 40 isolates exhibited thermophlic growth, 95 were found to be thermotolerant.
Most of the isolates were able to grow with pH range 6.5-8.5 and 0-5 % of NaCl.

All thermophilic aerobic endospore-forming isolates were identified
based on their 16S rRNA gene analysis. BLAST results for
the isolates, based on 16S rRNA gene sequences for identifcation
of the closest relatives in the GenBank database indicate that they
all belong to Clostridium-Bacillus subphylum, group of Bacillus-like
genera distributed in genera Anoxybacillus, Bacillus, Brevibacillus,
Geobacillus, Paenibacillus, Sporosarcina, Ureibacillus
and Thermoactinomyces (Table 3).

Representatives of the genera
Geobacillus and Anoxybacillus are the most distributed obligate
thermophiles in the studied hot springs. All isolates from the hot
springs that belonged to the genus Bacillus were thermotoleant
microorganisms among which B. licheniformis appeared as the
dominating species. Studied springs demonstrated significantly
lower content of species belonged to genera Brevibacillus, Ureibacillus
Paenibacillus, Thermoactinomyces and Sporosarcina. Some
bacilli sequences shared less than 96-97% identity with their closest
match in GenBank, indicating that the Armenian geothermal
springs harbour novel bacilli species. While these results are important
for further taxonomic work, positive results on hydrolitic
activities are indicative of potential application of these bacterial
cultures.

Number of thermophilic bacilli species belonging to the genera
Bacillus, Geobacillus and Anoxybacillus have been isolated from
different geothermal springs and reported as thermostable amylase,
lipase and proteinase producers (Raddadi et al, 2015; DeCastro
et al., 2016). Amylases are among the most important commercial
enzymes, comprising 25 % of the total enzyme market and
used in areas like manufacturing high fructose-containing syrups,
fermentation of starch to ethanol, treatment of starch processing
waste water (Elleuche, Antranikian, 2013). Lipases have a wide
area of usage in detergent, food, bio-resolution of pharmaceuticals,
agrochemicals, bioremediation, cosmetics and perfumery industry
(Sharma et al., 2013). Proteases, which constitute the 60 % of the
global enzyme market, have a wide area of usage in various sectors
like detergents, leather, food, cosmetics, medicine and medical diagnosis
(Sinha, Khare, 2013).

Therefore, the potential of bacilli species, as candidates for commercial
production thermostable hydrolases should be considered.
Taken into account this information, amylolytic, proteolytic, and
lipolytic enzyme activities of the isolates were detected using a
plating technique (Fig. 2). In total 65 strains (almost half of the
isolates) showed good hydrolysis of the starch substrate, as indicated
by the production of clear zones on the plate. In total 52
lipase and more than 30 thermophilic protease producers were selected
among the obtained isolates. Some of isolates which were
the members of the genus Bacillus, Anoxybacillus and Geobacillus
were lipase and amylase producers. According to the petri
dish enzyme assays, a large number of strains have the production
capacity of more than one enzyme. Some of the isolates (mainly
representative of genus Bacillus), were producers of the three enzymes
together.

Our results revealed the phylogenetic diversity of a large number of
thermophilic bacilli in Armenian geothermal hot springs. As part
of the microbiota, thermophilic bacilli presumably make significant
contributions in the biogeochemical cycles of the springs under
extreme temperature con-di¬tions. The results obtained show
the importance of further investigation of the phy¬lo¬ge¬netic
diversity of microbes in geothermal springs to discover and
iso¬late new thermophilic species. The ability of amylase, lipase
and protease production of isolates indicates their potential to use
in biotechnology as valuable enzyme producers. In summary, obtained
isolates from Armenian geothermal springs demonstrate the
diversity of thermophilic bacilli with hydrolytic activity inhabiting
these springs.

Acknowledgment

The work was supported by Research Grant from State Committee
of Science, Ministry of Education and Science of Armenia, to
HP (15T-1F399), Armenian National Science and Education Fund
based in New York, USA, to HP (ANSEF-NS-microbio 3362 and
4676) and grant from the Norwegian Cooperation Program in
Higher Education with Eurasia (CPEA-2011/10081).